1. Introduction

Nucleosomes consisting of approximately 146 base pairs (bp) of DNA wrapped around a histone octamer are the fundamental structural units of chromatin in metazoans (1, 2). The translational positioning of nucleosomes along DNA is implicated in profoundly influencing gene expression (3-6). Thus, defining the nucleosome positioning and occupancy is critical to understand the mechanisms of regulation of transcription by chromatin.

Nucleosome structure is resistant to microccocal nuclease (MNase) digestion, leaving a footprint of about 150bp that reflects the position of a nucleosome (7). Therefore, determining the boundaries of these footprints can indicate the positions of nucleosomes in the genome. Since the genomic sequences of most model organisms are already available, sequencing a short tag from DNA at each end of the nucleosome is sufficient to determine its position in the genome. Thus the next generation sequencing techniques are perfectly suited for this purpose (8).

We have generated genome-wide maps of nucleosome positions in both resting and activated human CD4+ T cells by direct sequencing of nucleosome ends using the Illumina Genome Analyzer Platform (MNase-Seq) (9). As the next generation sequencing techniques improve, the capacity and cost of sequencing become lower. For example, one sequencing run on the Illumina Genome Analyzer II can produce 100 to 200 millions of sequencing reads, which is sufficient to reach a 10x coverage for all nucleosomes in the human genome.

We describe two different methods to prepare nucleosome templates used for sequencing. One is digestion of native chromatin and the other is digestion of formaldehyde-crosslinked chromatin by MNase. The native nucleosome protocol works well to reveal stable nucleosome structure and avoid crosslinking of non-histone proteins; the crosslinking protocol may stabilize “unstable” nucleosomes but may also stabilize non-nucleosome structure that is resistant to MNase digestion.

Pellet the nuclei by spinning at 2000rpm (350×g) for 5min at 4°C. Wash the nuclei with 1 ml MNase digestion buffer, spin down at 2000rpm for 5 min at 4°C and resupend the pellet in 800μl of the same buffer (at a concentration of 10-20 million nuclei per ml). Adjust the final Ca2+ concentration to 1mM with 1M CaCl2.

Aliquot the nuclei suspension into eight tubes (100μl each), to which 0, 0.01, 0.03, 0.05, 0.1, 0.3, 0.5, and 1 units of MNase are added, respectively. Incubate the reaction mixture at 37°C for 5min (Note 1), then stop the reaction by adding 150μl of Stop Buffer.

Incubate the mixture at 65°C for 6 hrs or overnight.

Extract the mixture using an equal volume of phenol/chloroform.

Add 20μg of Glycogen from a stock solution to the aqueous phase, precipitate the DNA with 750μl of ethanol and 75μl of 3M NaAc, pH5.3, wash the DNA pellet once with 750μl of ice-cold 70% ethanol, and dissolve the pellet in 30μl of 1X TE buffer.

Try 18 cycles first, check 2.5 μl of product on 1.8% gel. If the band is not clearly visible, do 3 more cycles. Check again.

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Purify the amplified products on 2.5% agarose gel.

Excise the band near 220 bp and purify the DNA using Qiagen gel extraction kit. Measure the DNA concentration using Qubit fluorometer (Note 4).

3.3. Sequencing and data analysis

Purified DNA is used directly for cluster generation and sequencing analysis on Illumina IIX Genome Analyzer following manufacturer protocols.

Data Analysis: Sequenced reads of mostly 25 bp are obtained using the Illumina Analysis Pipeline. All reads are mapped to the human genome (hg18) or other reference genomes and all uniquely matching reads are retained. Nucleosome profiles are obtained by applying a scoring function to the sequenced reads. A sliding window of 10 bp is applied across all chromosomes and at each window all reads mapping to the sense strand 80 bp upstream of the window and reads mapping to the antisense strand 80 bp downstream of the window contribute equally to the score of the window.

Footnotes

1Incubate the reaction for 8-10min at 37°C for formadehydecrosslinked nuclei.

2The E-Gel® agarose gel electrophoresis system is a complete bufferless system for agarose gel electrophoresis of DNA samples. It provides fast, safe, consistent, high-resolution electrophoresis and minimizes sample contamination. E-Gel® EX pre-cast agarose gels are generally used gels which contain a proprietary fluorescent nucleic acid stain with high sensitivity, allowing: (1)Detection of down to 1 ng/band of DNA, (2)Compatibility with blue light transillumination to dramatically reduce DNA damage, (3)Easy opening of cassette with gel knife. If this system is not available, traditional gel purification methods can be used, but cross-contamination may result.

4Dissolve gel slices at room temperature with frequent mixing, but not at elevated temperature. This helps to preserve AT-rich DNA that can be easily denatured at higher temperature and could then be lost at the column binding step.

5Warm up EB at 65°C. Ensure that the EB is dispensed directly onto the QlAquick membrane for complete elution of bound DNA.

6NEVER run PCR-amplified samples of Step 7 with the linker-ligated products of Step 5 together on the same gel because the latter can be contaminated.